Isolation, Purification and Characterization of Nucleoids from <i>Synechococcus elongatus</i>PCC 7942

The genomic DNA of bacteria is highly compacted in one or a few bodies known as nucleoids. In order to understand the overall configuration and physiological activities of the cyanobacterial nucleoid under various growth conditions and the role(s) of each nucleoid protein in clock function, thylakoid membrane-associated nucleoids from the Synechococcus elongatus (se) PCC 7942 strain were isolated and purified in presence of spermidine at low salt concentrations by sucrose density gradient centrifugation. The sedimentation rates, protein/DNA composition and microscopic appearances as well as variation in structural components of clock proteins from the isolated nucleoids were compared under identical conditions. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The nucleoid structure in the wild type was more tightly compacted than that in the KaiABC mutant strain. Western immunoblot analyses revealed that the KaiC was associated with the nucleoid fraction whereas maximum KaiA was localized in the cytosolic fraction, supposedly in association with the translation machinery.

Dps Is a Stationary Phase-Specific Protein of <i>Escherichia coli</i>Nucleoid

Bacterial genomic DNA is highly organized into one or few compacted bodies known as nucleoid, which is composed of DNA, RNA and several DNA-binding proteins. These DNA-binding proteins require essential alterations in their expression during stationary phase of growth in order to re-spond to stressful environmental conditions. Dps (DNA-binding protein from starved cells) is one of such DNA-binding proteins, which accumulates most when E. coli cells reach to the stationary phase. Here, we have characterized Dps protein under various growth phases. Immunofluorescent microscopic observation reveals that Dps plays a key role in final round of genome compaction during the stationary phase. Similar results are also obtained by Western immunoblot analysis, after quantification of Dps protein from the exponential phase and early stationary phase nucleoid bound fractions, separated by sucrose density gradient centrifugation. Our results support the conclusion that Dps occupies more than half of the stationary phase nucleoid in E. coli.

Features of Structurization at Participation of Guanidine Groups of Arginine in Life Cycle in Population of E. coil

The purpose of the given work was the experimental analysis of features of Arg-X proteolysis in proteom of supramolecular structures of bacterial cells during their life cycle. The basic attention was devoted to relaxation of Arg-X sites of proteom in association with the evolutionary significance ofArg-rich histones in the eukaryotic kingdom. These properties were not studied in the prokaryotes. Cells ofE. coli were grown to the stationary phase, collected by centrifugation and washed. All cells were taken over from 50 min to 430 min at intervals of 20 min and were preserved in glycerol. The supramolecular structures were fractionated from bacterial cells by increasing ionic strength of solution. The Arg-Xactivity was assessed by cleavage of Arg-Xbonds in the arginine-enriched protein protamine in all cell fractions. We have shown that during the stationary phase in the life cycle of E. coli, there are a high continuous activity of the Arg-X processing at the level of＂cytoskeleton＂ of the cell and bright cyclic activity in the cytoplasm.

类核环形结构在耐辐射球菌电离辐射抗性中不起主要作用

近来基于透射电子显微镜研究的论点“耐辐射球菌(Deinococcus radiodurans)R1类核致密的环形结构是其极端辐射抗性的关键因素”已引起广泛争议.本文在以前研究基础上系统地比较了野生型R1菌株、pprI(DR0167)基因突变株YR1及pprI基因功能补偿株在电离辐射前后的类核结构的形态差异.荧光显微镜观察结果显示:(ⅰ)具有超强电离辐射抗性的野生型菌株R1细胞类核主要呈现紧凑的环形结构,而同样有环形类核结构的pprI突变株YR1细胞却对辐射非常敏感;(ⅱ)2个pprI基因功能补偿株,YR1001(pprI全基因功能补偿株)类核绝大多数呈现絮乱松散的不规则形态,YR1002(pprI部分基因功能补偿株)呈现约60%左右的环形类核结构,这两个菌株都具有与野生型R1同样的电离辐射抗性;(ⅲ)另一pprI部分基因功能补偿株YR1004(敲除pprI基因3′端333个碱基对)对电离辐射极其敏感,其约60%左右的细胞含有环形的类核结构.上述结果表明,耐辐射球菌类核的环形结构在辐射极端抗性中不起主要作用.

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

The chloroplasts genome （plastome） occurs at high copy numbers per cell. Several chloroplast genome copies are densely packed into nucleoprotein particles called nucleoids. How genome packaging occurs and which proteins organize chloroplast nucleoids are largely unknown. Here, we have analyzed the Chlamydornonas reinhardtii homolog of the bacterial architectural DNA-binding protein HU, the histone-like protein HLP. We show that the Chlarnydornonas HLP protein is targeted to chloroplasts and associates with nucleoids. Knockdown of HLP gene expression by RNA interference （RNAi） alters the structure of chloroplast nucleoids and appears to reduce the level of compaction of chloroplast DNA. Unexpectedly, also chloroplast genome copy numbers are significantly decreased in the RNAi strains, suggesting that, in addition to its architectural role in nucleoid formation, the HIP protein is also involved in chloroplast genome maintenance.

Growth phase dependent changes in the structure and protein composition of nucleoid in Escherichia coli

The genomic DNA of bacteria is highly compacted in a single or a few bodies known as nucleoids. Here, we have isolated Escherichia coli nucleoid by sucrose density gradient centrifugation. The sedimentation rates, structures as well as pro- tein/DNA composition of isolated nucleoids were then compared under various growth phases. The nucleoid structures were found to undergo changes during the cell growth; i. e., the nucleoid structure in the stationary phase was more tightly com- pacted than that in the exponential phase. In addition to factor for inversion stimulation （Fis）, histone-like nucleoid structuring protein （H-NS）, heat-unstable nucleoid protein （HU） and integration host factor （IHF） here we have identified, three new can- didates of E. coli nucleoid, namely DNA-binding protein from starved cells （Dps）, host factor for phage QJ3 （Hfq） and sup- pressor of taC phenotype A （StpA）. Our results reveal that the major components of exponential phase nucleoid are Fis, HU, H-NS, StpA and Hfq, while Dps occupies more than half of the stationary phase nucleoid. It has been known for a while that Dps is the main nucleoid-associated protein at stationary phase. From these results and the prevailing information, we propose a model for growth phase dependent changes in the structure and protein composition of nucleoid in E. coli.

Does the eclipse limit bacterial nucleoid complexity and cell width?

Cell size of bacteria M is related to 3 temporal parameters:chromosome replication time C,period from replication-termination to subsequent division D,and doubling time t.Steady-state,bacillary cells grow exponentially by extending length L only,but their constant width W is larger at shorter t‘s or longer C's,in proportion to the number of chromosome replication positions n(?C/t),at least in Escherichia coli and Salmonella typhimurium.Extending C by thymine limitation of fast-growing thyA mutants result in continuous increase of M,associated with rising W,up to a limit before branching.A set of such puzzling observations is qualitatively consistent with the view that the actual cell mass(or volume)at the time of replication-initiation Mi(or Vi),usually relatively constant in growth at varying t0s,rises with time under thymine limitation of fast-growing,thymine-requiring E.coli strains.The hypothesis will be tested that presumes existence of a minimal distance lmin between successive moving replisomes,translated into the time needed for a replisome to reach lmin before a newreplication-initiation at oriC is allowed,termed Eclipse E.Preliminary analysis of currently available data is inconsistent with a constant E under all conditions,hence other explanations andways to test themare proposed in an attempt to elucidate these and other results.The complex hypothesis takes into account much of what is currently known about Bacterial Physiology:the relationships between cell dimensions,growth and cycle parameters,particularly nucleoid structure,replication and position,and themode of peptidoglycan biosynthesis.Further experiments arementioned that are necessary to test the discussed ideas and hypotheses.